PATHOGENESIS OF PARAMYXOVIRUSES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0230904
• Project No.: PEN04497
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2016

Project Director
Schmitt, AN.

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802

Performing Department
Veterinary & Biomedical Sciences

Non Technical Summary
The paramyxoviruses comprise a large group of negative-strand RNA viruses, many of which are significant causes of disease both in humans and in agriculturally important animals. Paramyxoviruses, like other enveloped viruses, spread from one host to another in the form of particles. The particles bud from the plasma membranes of virus-infected cells, allowing the transfer of infectious material to uninfected cells. The process of particle assembly and release is one of the least-understood aspects of the paramyxovirus lifecycle. Improved knowledge of the particle formation process will contribute to the development of effective antiviral strategies aimed at blocking the late steps of paramyxovirus replication. Paramyxovirus particle formation is driven by the viral M (matrix) proteins. One of the key protein interactions mediated by M proteins involves the viral nucleocapsid protein, NP. NP protein functions to bind and encapsidate the viral genomic RNA. The interaction between the viral M protein (located at virus budding sites) and NP protein (complexed with viral RNA) is the driving force that allows for the selective packaging of viral genomes into budding virions. Here, experiments are proposed that will characterize the binding interface between paramyxovirus M and NP proteins. This will set the stage for future efforts aimed at disrupting the M-NP interaction. Impaired M-NP interaction would be devastating to the virus, because viruses would no longer be able to incorporate their genomes into the particles that spread the infection. Experiments are also proposed that will characterize interactions between viral M proteins and two different host proteins: 14-3-3 and AP-3. This will facilitate the development of antiviral strategies aimed at blocking the interactions between M proteins and host proteins that are critical for proper virus assembly and particle formation.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
722	4030	1101	85%
311	4030	1101	15%

Knowledge Area
311 - Animal Diseases; 722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1101 - Virology;

Keywords

virus

paramyxovirus

virus assembly

virus budding

virus particle

antiviral

Goals / Objectives
The overall goal of this project is to define new targets for therapeutic intervention against paramyxoviruses, specifically targeting viral protein interactions that normally function to allow virus particle assembly. Specific objectives: 1. Define binding interfaces between paramyxovirus NP and M proteins that allow for the incorporation of genomes into budding particles. 2. Assess the consequences of altered NP-M interaction using recombinant viruses. 3. Characterize the functions of M-interacting host factors in paramyxovirus replication.

Project Methods
Objective 1: Amino acid residues within paramyxovirus NP proteins that are important for interaction with the viral M proteins will be defined. NP protein sequences will be fused to Renilla luciferase, and the modified luciferase proteins will be expressed in cells together with the viral M proteins. Virus-like particles will be collected from the cells, and incorporation of the modified luciferase into particles will be quantified. Mutagenesis of the viral M and NP proteins will allow further definition of critical binding interfaces. Objective 2: Recombinant viruses will be generated with altered M proteins that either impair or enhance the interaction with NP protein. Viruses will be characterized using standard molecular virology techniques, including growth curve assays, measurements of viral protein synthesis, measurements of viral particle formation, polypeptide composition of particles, virion infectivity, and microscopic examination of virions as well as virus-infected cells. Obective 3: Recombinant viruses will be generated with altered M proteins that fail to interact with the cellular protein 14-3-3. These viruses will be examined using a variety of molecular virology techniques, as described for Objective 2.

Progress 10/01/12 to 09/30/16

Outputs
Target Audience:Biotechnology companies, companies interested in antiviral drug development, companies interested in drug delivery or protein delivery technologies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The subproject on trafficking and assembly of Hendra virus M, F, and G proteins was carried out by a graduate student, Weina Sun, who successfully completed her PhD in 2015. The subproject on M-NP interactions that direct paramyxovirus genome packaging was carried out by a graduate student, Greeshma Ray, who successfully completed her PhD in 2016. The subproject on paramyxovirus M-interacting host factors was also carried out by Greeshma Ray. How have the results been disseminated to communities of interest?Results from the subproject on M-NP interactions that direct paramyxovirus genome packaging were published in the Journal of Virology, which is widely regarded as a top-tier virology journal. Results from the subproject on Hendra virus assembly were also published in the Journal of Virology. Results from all subprojects have been presented at scientific conferences and invited speaking engagements, including the American Society for Virology 2012, 2013, 2015, and 2016 meetings, a seminar presented at Northwestern University in August of 2015, the "Living with Our Viromes" PSU Summer Symposium in Molecular Virology in 2016, and a "Germinator" technology forum hosted by GSK in 2016. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. M-NP interactions that direct paramyxovirus genome packaging. We discovered that short, DLD-containing sequences near the C-terminal ends of PIV5 and Nipah virus nucleocapsid proteins are sufficient to direct a foreign protein into budding particles. This finding provides mechanistic insight into how paramyxoviruses incorporate NP-encapsidated RNA genomes into virus particles. In addition, these results provide the fundamental basis for development of a new strategy aimed at incorporation of foreign proteins into virus-like particles, for subsequent delivery to target cells. This work has been recently published in the Journal of Virology. This work also forms the basis of a provisional patent application that was filed in December of 2015. 2. M-NP interactions that direct paramyxovirus genome packaging. We described a novel approach to engineering compatibilities between M and NP proteins of different paramyxoviruses. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggests a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions, but with ?important variations contained within DLD-like sequences that play key roles in defining M/NP ?protein compatibilities. This work has been recently published in the Journal of Virology. 3. Paramyxovirus M-interacting host factors. We previously identified a host factor, angiomotin-like 1 (AmotL1) that binds to the PIV5 and mumps virus M proteins. During this reporting period, we found that binding is selective to AmotL1 and does not extend to the other members of the angiomotin family - Amot or AmotL2. Consistent with this, we found that PIV5 budding could be inhibited by AmotL1-derived, M-binding polypeptides, but could not be inhibited by polypeptides derived from Amot or AmotL2. We propose that AmotL1 recruitment serves to indirectly recruit Nedd4-like proteins to PIV5 assembly sites, using the same interactions that directly recruit Nedd4-like proteins to the assembly sites of Ebola virus and other enveloped viruses. Co-immunoprecipitation of Nedd4 with PIV5 M protein was observed only if AmotL1 was co-expressed as a bridging protein. Our results suggest a model in which paramyxoviruses such as PIV5 and mumps virus indirectly recruit ESCRT components for budding through AmotL1. 4. Trafficking and assembly of Hendra virus M, F, and G proteins. In a collaborative effort with Dr. Rebecca Dutch at the University of Kentucky, we identified F and G protein mutants that fail to assemble together with M protein to incorporate into budding VLPs. Incorporation of G protein into VLPs was found to be M-dependent, while F protein could induce particle formation even in the absence of M.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ray, G., Schmitt, P.T., and Schmitt, A.P. (2016). C-terminal DxD-containing sequences within paramyxovirus nucleocapsid proteins determine matrix protein compatibility, and can direct foreign proteins into budding particles
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sun, W., McCrory, T.S., Khaw, W.Y., Petzing, S., Myers, T., and Schmitt, A.P. (2014). Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes. J. Virol., 88, 13099-13110.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: El Najjar, F., Schmitt, A.P., and Dutch, R.E. (2014). Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production. Viruses. 6, 3019-3054.
• Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Johnson, J.B., Schmitt, A.P., and Parks, G.D. (2013). Point Mutations in the Paramyxovirus F Protein That Enhance Fusion Activity Shift the Mechanism of Complement-Mediated Virus Neutralization. J. Virol. 87, 9250-9259.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Biotechnology companies, companies interested in antiviral drug development, and companies interested in protein delivery technologies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The subproject on trafficking and assembly of Hendra virus M, F, and G proteins was carried out by a graduate student, Weina Sun, who successfully completed her PhD in 2015. The subproject on M-NP interactions that direct paramyxovirus genome packaging was carried out by a graduate student, Greeshma Ray, who is expected to complete her PhD in early 2016. The subproject on paramyxovirus M-interacting host factors also was carried out by Greeshma Ray. How have the results been disseminated to communities of interest?An article on the subproject focused on how M-NP interactions that direct paramyxovirus genome packaging was submitted for publication in the Journal of Virology, which is widely regarded as a top-tier virology journal. Results from all subprojects have been presented at scientific conferences and invited speaking engagements, including the American Society for Virology 2015 meeting, and a seminar presented at Northwestern University in August of 2015. What do you plan to do during the next reporting period to accomplish the goals?The subproject on trafficking and assembly of Hendra virus M, F, and G proteins is nearing completion, and the results will be published in a high quality peer-reviewed journal. The subproject on the role of angiomotin proteins in paramyxovirus budding is at approximately the midway point. Additional experiments will be conducted to define the functional importance of AmotL1-mediated Nedd4 recruitment to paramyxovirus budding sites. Results will be published in a high-quality, peer-reviewed journal. The subproject on paramyxovirus M-NP interactions has entered a new, more translational phase in which we plan to demonstrate the utility of paramyxovirus-like particles as protein delivery vehicles.

Impacts
What was accomplished under these goals? 1. M-NP interactions that direct paramyxovirus genome packaging: We discovered that short, DLD-containing sequences near the C-terminal ends of PIV5 and Nipah virus nucleocapsid proteins are sufficient to direct a foreign protein into budding particles. This finding provides mechanistic insight into how paramyxoviruses incorporate NP-encapsidated RNA genomes into virus particles. In addition, these results provide the fundamental basis for development of a new strategy aimed at incorporation of foreign proteins into virus-like particles (VLPs) for subsequent delivery to target cells. This work has resulted in an article that has been submitted for publication in the Journal of Virology. 2. M-NP interactions that direct paramyxovirus genome packaging: We described a novel approach to engineering compatibilities between M and NP proteins of different paramyxoviruses. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data supports a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions, but with important variations contained within DLD-like sequences that play key roles in defining M-NP protein compatibilities. This work has resulted in a manuscript that has been submitted for publication in the Journal of Virology. 3. Paramyxovirus M-interacting host factors: We previously identified a host factor, angiomotin-like 1 (AmotL1), that binds to the PIV5 and mumps virus M proteins. During this reporting period, we found that binding is selective to AmotL1 and does not extend to the other members of the angiomotin family: Amot or AmotL2. Consistent with this, we found that PIV5 budding could be inhibited by AmotL1-derived, M-binding polypeptides, but could not be inhibited by polypeptides derived from Amot or AmotL2. We propose that AmotL1 recruitment serves to indirectly recruit Nedd4-like proteins to PIV5 assembly sites, using the same interactions that directly recruit Nedd4-like proteins to the assembly sites of Ebola virus and other enveloped viruses. Co-immunoprecipitation of Nedd4 with PIV5 M protein was observed only if AmotL1 was co-expressed as a bridging protein. Our results support models in which paramyxoviruses, such as PIV5 and mumps virus, indirectly recruit endosomal sorting complexes required for transport (ESCRT) components for budding through AmotL1. 4. Trafficking and assembly of Hendra virus M, F, and G proteins: In a collaborative effort with Dr. Rebecca Dutch at the University of Kentucky, we identified F and G protein mutants that fail to assemble together with M protein to incorporate into budding VLPs. Incorporation of G protein into VLPs was found to be M-dependent, while F protein could induce particle formation even in the absence of M.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Ray, G., Schmitt, P.T., and Schmitt, A.P. (2015). C-terminal DxD-containing sequences within paramyxovirus nucleocapsid proteins determine matrix protein compatibility, and can direct foreign proteins into budding particles.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Stake holders, biotechnology companies, and companies interested in antiviral drug development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The subproject on M protein interaction with AP-3 was carried out by two graduate students: Thomas McCrory successfully completed his PhD in 2012; and Weina Sun is expected to complete her PhD in early 2015. The subproject on trafficking and assembly of Hendra virus M and F proteins was also carried out in its entirety by Weina Sun. How have the results been disseminated to communities of interest? Results of the subproject on M protein interaction with AP-3 were published in the Journal of Virology, which is widely regarded as a top-tier virology journal. Results of the subproject on complement-mediated neutralization of paramyxovirus particles also were published in the Journal of Virology. Results of all subprojects have been presented at scientific conferences and invited speaking engagements, including a seminar presented at the University of Alabama at Birmingham in March of 2014. What do you plan to do during the next reporting period to accomplish the goals? The subproject on trafficking and assembly of Hendra virus M and F proteins is approximately at the midway point. Additional experiments will be conducted to define intracellular locations where M and F trafficking pathways intersect. Results will be published in a high-quality, peer-reviewed journal. A project characterizing paramyxovirus M-NP interactions is nearing completion, and the results will be published in a high-quality, peer-reviewed journal. A new subproject to characterize the role of angiomotin proteins in paramyxovirus budding has been initiated. We expect to advance our understanding of M-angiomotin interactions and their importance to virus budding during the next period.

Impacts
What was accomplished under these goals? 1. M protein interaction with AP-3. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production also was impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. 2. Trafficking and assembly and Hendra virus M and F proteins. In a collaborative effort with Dr. Rebecca Dutch at the University of Kentucky, we have identified F protein mutants that fail to be internalized from the cell surface. When co-expressed with M protein, these F mutants are not assembled into budding VLPs, whereas wt F protein assembles efficiently into VLPs. This result suggests that proper endocytic trafficking of F protein is necessary for its subsequent assembly with M protein for virus budding. 3. Complement-mediated neutralization of paramyxovirus particles. In a collaborative effort with Dr. Griffith Parks at the University of Central Florida, we have defined distinct mechanisms for complement-mediated neutralization of PIV5 and mumps virus particles. Using VLPs, we have demonstrated that both the F and HN glycoproteins of these viruses are targets for inactivation through complement.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sun, W., McCrory, T.S., Khaw, W.Y., Petzing, S., Myers, T., and Schmitt, A.P. (2014). Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes. J. Virol., 88, 13099-13110.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: El Najjar, F., Schmitt, A.P., and Dutch, R.E. (2014). Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production. Viruses, 6, 3019-3054.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Johnson, J.B., Schmitt, A.P., and Parks, G.D. (2013). Point mutations in the paramyxovirus F protein that enhance fusion activity shift the mechanism of complement-mediated virus neutralization. J. Virol., 87, 9250-9259.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Stake holders, biotechnology companies, companies interested in antiviral drug development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The subproject on M protein monobuiquitination was carried out by a graduate student, Megan Harrison, who was trained in all aspects of paramyxovirus molecular virology during the course of her work. She successfully completed her PhD in 2011. The subproject on M protein interaction with AP-3 was carried out by two graduate students. Thomas McCrory successfully completed his PhD in 2012. Weina Sun is expected to complete her PhD in 2014. How have the results been disseminated to communities of interest? The subproject on M protein monobuiquitination was carried out by a graduate student, Megan Harrison, who was trained in all aspects of paramyxovirus molecular virology during the course of her work. She successfully completed her PhD in 2011. The subproject on M protein interaction with AP-3 was carried out by two graduate students. Thomas McCrory successfully completed his PhD in 2012. Weina Sun is expected to complete her PhD in 2014. What do you plan to do during the next reporting period to accomplish the goals? The subproject on M protein interaction with AP-3 is nearing completion and will be published in a high-quality peer-reviewed journal. A project on trafficking and assembly of Henipavirus M and F proteins has been initiated in collaboration with Dr. Rebecca Dutch at the University of Kentucky, and we expect to advance our understanding of M-F assembly during the next period. A project to characterize paramyxovirus M-NP interactions is underway, and we expect to advance our understanding of this important interaction for virus assembly during the next period.

Impacts
What was accomplished under these goals? M protein monoubiquitination: Ubiquitin is important for the budding of many enveloped viruses, and monoubiquitination of viral proteins may serve as a connection between certain viral proteins and cellular ESCRT machinery. We demonstrated that PIV5 M protein is a target for ubiquitin attachment, and using mass spectrometry identified specific lysine residues that are the preferred sites for monoubiquitination. Cumulative removal of lysine residues from M protein altered its ubiquitination and impaired its budding function. Recombinant PIV5 with seven M protein lysine residues changed to arginine exhibited particle assembly defects including mislocalization of M protein, reduced particle release, and poor infectivity of the released particles. These results suggest that PIV5 M protein monoubiquitination contributes to M protein trafficking, assembly, and particle release. M protein interaction with AP-3: We have defined an interaction between Henipavirus M proteins and the beta subunits of AP-3 adapter complexes. Binding was mapped to a short serine-rich, acidic region of AP3B1. Budding and release of Henipavirus-like particles was inhibited by siRNA-mediated depletion of AP3B1, and was also inhibited by expression of short, M-binding, AP3B1-derived polypeptides. These results define AP3B1 as an important host factor required for proper assembly and release of Henipavirus particles.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Harrison, M.S., Schmitt, P.T., Pei, Z., and Schmitt, A.P. (2012). Role of ubiquitin in PIV5 particle formation. J. Virol. 86, 3474-3485. PMC3302527.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Racicot, K., Schmitt, A., and Ott, T. (2012). The myxovirus?resistance protein, MX1, is a component of exosomes secreted by uterine epithelial cells. Am. J. Reprod. Immunol., 67, 498-505. PMC in progress.